title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.
abstract: The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.
authors.affiliation: Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland, UK. ltrinkle@uottawa.ca
authors.forename: Laura
authors.initials: L
authors.lastname: Trinkle-Mulcahy
authors.forename: Séverine
authors.initials: S
authors.lastname: Boulon
authors.forename: Yun Wah
authors.initials: YW
authors.lastname: Lam
authors.forename: Roby
authors.initials: R
authors.lastname: Urcia
authors.forename: François-Michel
authors.initials: FM
authors.lastname: Boisvert
authors.forename: Franck
authors.initials: F
authors.lastname: Vandermoere
authors.forename: Nick A
authors.initials: NA
authors.lastname: Morrice
authors.forename: Sam
authors.initials: S
authors.lastname: Swift
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Angus
authors.initials: A
authors.lastname: Lamond
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D030562:Databases, Protein; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008863:Microspheres; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012685:Sepharose; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D020543:Proteome; D011993:Recombinant Fusion Proteins; C506234:USP9X protein, human; D049452:Green Fluorescent Proteins; D012685:Sepharose; D043222:Ubiquitin Thiolesterase